Molecular regulation of cholesterol concentration in cell membranes

• Applicant: Professor Dr. Stefan Raunser
• Subject Area: Structural Biology
• Term: from 2008 to 2013
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 60803023

Cholesterol plays many important roles in mammalian cells. It is a major component of the plasma membrane and membranes of various organelles. Mammalian cells acquire cholesterol by de novo synthesis in the endoplasmic reticulum (ER) and by endocytosis of lipoproteins. Both the uptake and the synthesis of cholesterol are tightly regulated by feedback mechanisms. Malfunction of this regulation as found in familial hypercholesterolemia is associated with the early stages of atherosclerosis, and high levels of cholesterol in these cells can lead to cell death in late stages of atherosclerosis.The membrane spanning proteins Insig (original name “insulin induced gene protein”), sterol regulatory element binding protein (SREBP) and SREBP cleavage-activating protein (Scap) are the core proteins in the regulation of cholesterol synthesis. At high cholesterol levels in the membrane Insig, SREBP and Scap form a macromolecular complex. Whereas the structure of the SREBP transcription factor domain has been reported, structural information of the complete SREBP protein, Insig, Scap or the Insig/Scap/SREBP complex is missing. Structural information will be required to understand how these different proteins interact in the regulation of cholesterol synthesis. Besides biochemical and functional analysis, the primary objective of this proposal is therefore to determine the three-dimensional structures of the mammalian sterol regulatory element binding protein (SREBP), the mammalian SREBP cleavage-activating protein (Scap) and the mammalian insulin induced gene protein (Insig) by electron crystallography of two-dimensional crystals. In parallel the 3D structure of the mammalian Insig/Scap/SREBP complex and its homolog ins1/scp1/sre1 from Schizosaccharomyces pombe will be determined by single particle cryo-electron microscopy and its individual components will be localized within this structure by immunolabeling.

• DFG Programme: Independent Junior Research Groups
• Major Instrumentation: Proteinreinigungssystem
• Instrumentation Group: 3190 Sonstige Geräte der Klinischen Chemie und Molekularbiologie